Published August 29, 2016
Review
JCB: Spotlight
At the crossroads of autophagy and infection:
Noncanonical roles for ATG proteins in
viral replication
Tina Solvik1,2,3 and Jayanta Debnath1,2
of Pathology, 2Helen Diller Family Comprehensive Cancer Center, and 3Biomedical Sciences Graduate Program, University of California, San Francisco,
San Francisco, CA 94143
Autophagy-related (ATG) proteins have increasingly
demonstrated functions other than cellular self-eating. In
this issue, Mauthe et al. (2016. J. Cell Biol. http​://dx​.doi​
.org​/10​.1083​/jcb​.201602046) conduct an unbiased
RNA interference screen of the ATG proteome to reveal
numerous noncanonical roles for ATG proteins during
viral infection.
Macroautophagy (autophagy) involves the formation of a double-membrane organelle called the autophagosome, which sequesters cytoplasmic components that are degraded upon its
fusion with the lysosome (Stolz et al., 2014; Kaur and Debnath,
2015). Studies in yeast have identified >30 autophagy-related
(ATG) proteins, many of which have identified mammalian
orthologues. The canonical process of autophagy begins with
initiation at the phagophore assembly site, mediated by the
UNC51-like kinase (ULK) complex, which is composed of
the ATG proteins ULK1/2, ATG13, FIP200, and ATG101. The
ULK complex supports the activation of a class III phosphatidylinositol 3-kinase complex consisting of Beclin 1 (ATG6),
ATG14, and VPS34 to produce phosphatidylinositol triphosphate, which serves as the initial membrane mark recognized
by early autophagic effector proteins. Phagophore membrane
expansion is mediated by the two ubiquitin-like conjugation
systems involving multiple ATG proteins that ultimately lead
to the lipidation of microtubule-associated protein 1 light chain
3 (LC3), the mammalian orthologue of ATG8. LC3 is recognized by autophagy cargo receptors that promote the selective
capture and engulfment of proteins, organelles, or microbes.
The closed autophagosome is then trafficked to and fuses with
the lysosome to degrade its cargo (Stolz et al., 2014; Kaur and
Debnath, 2015). ATG proteins traditionally have been studied
for their roles in autophagosome formation and maturation, but
we have recently begun to appreciate that they may have pleiotropic roles beyond autophagy (Bestebroer et al., 2013). In this
issue, through a comprehensive functional screen for ATG proteins in the control of viral replication, Mauthe et al. highlight
the importance of the nontraditional functions of ATG proteins
in the host viral response.
In a tour-de-force study examining multiple models of
viral infection, Mauthe et al. (2016) interrogated the individual
roles of ATG proteins in the replication of herpes simplex virus
Correspondence to Jayanta Debnath: [email protected]
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 214 No. 5 503–505
www.jcb.org/cgi/doi/10.1083/jcb.201608032
type 1, mouse hepatitis virus (MHV), vaccinia virus (VaV),
semliki forest virus, encephalomyocarditis virus, and influenza
A virus. The authors generated a curated siRNA library to target
all ATG proteins individually as well as functionally redundant
groups concurrently to evaluate how they promote the replication of these six viruses in two model cell lines (HeLa and
U2OS). Indeed, their screen reveals that unconventional functions of ATG proteins are widespread; overall, 16 out of the
44 tested ATG proteins (36%) appear to have unconventional
functions. Rarely does knockdown of individual ATG genes result in concordant effects on viral replication, arguing against a
general role for autophagy. Moreover, the authors fail to identify any single ATG protein that regulates viral replication in all
of the tested viruses; rather, specific ATG genes influenced the
replication of each virus. Remarkably, most of the ATG proteins
show a beneficial role for VaV replication in U2OS cells yet
negatively impact VaV replication in HeLa cells. Overall, the
effect of ATG loss of function on viral replication is exquisitely
ATG specific, virus specific, and cell line specific.
To demonstrate functions of ATG proteins independent
of autophagy, the authors scrutinized ATG13 and FIP200, two
ULK complex proteins that initiate formation of the autophagosome, and found that these proteins exert effects on viral
replication distinct from the effects of other ULK complex
components. Mauthe et al. (2016) demonstrate that depletion
of ATG13 and FIP200 reduces the replication of two picornaviruses, encephalomyocarditis virus and coxsackievirus (CV)
B3, whereas the other ULK complex components promote
their replication. Furthermore, a principal upstream regulator of ULK complex activity, mammalian target of rapamycin
complex 1, showed no changes in activity during infection or
knockdown of the ULK complex components. Altogether, this
evidence indicates that ATG13 and FIP200 act independently
of the ULK complex to modify picornaviral replication outside
the context of autophagy.
Although the precise mechanism of this unconventional
function of ATG13 and FIP200 remains to be elucidated, Mauthe et al. (2016) uncovered that these proteins control viral replication as opposed to viral entry or viral translation. However,
ATG13 and FIP200 do not interact with any CV proteins or affect the morphology of CV replication structures, suggesting
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THE JOURNAL OF CELL BIOLOGY
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© 2016 Solvik and Debnath This article is distributed under the terms of an Attribution–
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JCB
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Published August 29, 2016
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JCB • Volume 214 • Number 5 • 2016
no effect on M. tuberculosis infection in vivo. Instead, ATG5
prevents accumulation of polymorphonuclear cells in the lungs,
thereby controlling inflammation and M. tuberculosis replication (Kimmey et al., 2015).
During Brucella abortus infection, multiple ATG proteins
implicated in the early steps of autophagosome initiation, such as
ULK1 and Beclin 1, are involved in the formation of a B. abortus–containing vacuole (BCV) with autophagic features (aBCV),
which promotes bacterial proliferation. Double-membrane crescents reminiscent of the autophagosome engulf BCVs late in
infection but do not degrade or clear the bacteria. Instead, ULK1,
Beclin 1, and class III phosphatidylinositol 3-kinase activity are
all required for the conversion of BCVs into aBCVs. However,
the ATG conjugation pathways involved in autophagosome
elongation play no role in aBCV formation. Furthermore, these
aBCVs facilitate bacterial release and infection of neighboring
cells, demonstrating that very specific components of autophagosome initiation promote B. abortus infection (Starr et al., 2012).
Moreover, the lipidated form of LC3 (LC3-II) is essential for autophagosome formation and crucially interacts with
cytoplasmic cargo to be degraded. However, during MHV
infection, unlipidated LC3 (LC3-I) promotes viral replication in double-membrane vesicles (DMVs). The similarity of
DMVs to autophagosomes prompted speculation that classical
autophagy controlled MHV infection. However, ATG5 does
not affect infection, and only LC3-I colocalizes with MHV
DMVs, suggesting that canonical autophagy is not involved
(Zhao et al., 2007). Further investigation revealed that two
proteins in the ER-associated degradation pathway interact
with LC3-I and MHV replication translation complexes, that
MHV DMVs are of ER origin, and that LC3-I, but not LC3-II,
is required for MHV replication (Reggiori et al., 2010). Remarkably, these LC3-I–positive DMVs represent another
LC3-presenting membrane distinct from the canonical double-membrane autophagosome as well as the LC3-II–positive
single-membrane phagosome in LAP.
Lastly, during mouse norovirus (MNV) infection, IFN-γ–
activated phagocytes use ATG5-ATG12/ATG16L1 to inhibit
formation of the viral replication complex, thus restricting infection. This ATG complex typically promotes LC3 lipidation,
resulting in expansion of the autophagosome. Based on the requirement for ATG5 in IFN-γ–mediated inhibition of MNV replication, autophagy was initially suggested as the mechanism
of host defense. However, IFN-γ has no effect on autophagy in
phagocytes, and inducing or inhibiting autophagy does not alter
MNV replication. Instead, only ATG5-ATG12/ATG16L1 is required for MNV replication inhibition, and ATG16L1 localizes
to the MNV replication complex. ATG5-ATG12/ATG16L1 is
required for IFN-γ–mediated suppression of MNV polymerase
expression and, therefore, replication as a whole, an example
of multiple ATG proteins functioning to inhibit viral infection (Hwang et al., 2012).
Ultimately, the discovery of widespread autophagy-independent functions of ATG proteins marks a significant turning
point as well as reinforces an important caveat for the field.
Going forward, researchers must use caution when attributing
a phenotype to canonical autophagy based on the genetic analysis of a single ATG protein. The experiments in Mauthe et al.
(2016) poignantly illustrate the need to functionally test multiple ATG proteins when characterizing a phenotype to ascertain
a general role for autophagy as opposed to a distinct function
mediated by individual ATG proteins.
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the proteins have an indirect effect on viral replication. RNA
sequencing analysis demonstrates that ATG13 and FIP200 affect genes predominantly involved in transcriptional regulation
during CV infection. Future studies expanding on these results
are needed to dissect the exact mechanism by which ATG13 and
FIP200 reduce picornaviral replication.
Autophagy has inexorably been linked to the host cellular
response to diverse intracellular pathogens. Indeed, autophagy
frequently serves as an innate mechanism that sequesters or
clears offending pathogens from the host cell, termed xenophagy. In response to infection, pathogen- and damage-associated
molecular patterns stimulate pattern recognition receptors,
which then activate ATG proteins to induce autophagy or promote recognition of the microbe by the LC3-positive autophagosome (Deretic et al., 2015). Nevertheless, the autophagy
trafficking process can be subverted by pathogens that have
adapted to avoid or use autophagy to their own benefit (Deretic
and Levine, 2009). One example is poliovirus, which utilizes
LC3 to promote its nonlytic viral release, potentially by the
fusion of LC3-positive vesicles containing poliovirus with the
plasma membrane (Bird et al., 2014).
Interestingly, this last example broaches how ATG proteins, either as individual proteins or in groups, may enable unconventional functions distinct from the canonical process of
autophagy. This can include entirely novel functions or ATG
processes that use only a subset of the core components (Bestebroer et al., 2013). Before Mauthe et al. (2016), a comprehensive, unbiased analysis of the entire ATG proteome to uncover
the autophagy-independent functions of ATG proteins during
infection had not been undertaken. Nevertheless, other studies
have highlighted some of the emerging noncanonical roles of
ATG proteins during microbial pathogenesis. A well characterized example is LC3-associated phagocytosis (LAP), in which
a select group of ATG proteins promotes maturation and degradation of the phagosome. Upon toll-like receptor-triggered
phagocytosis, Beclin 1 is recruited to the phagosome, where it
induces production of phosphatidylinositol triphosphate and localization of LC3, causing fusion with the lysosome (Sanjuan
et al., 2007). Although LAP requires several ATG proteins—
Beclin 1, LC3, ATG5, and ATG7, among others—it is mechanistically distinct from autophagy as it does not use the ULK
complex that initiates canonical autophagy. In addition, LAP
utilizes proteins such as Rubicon, which is inhibitory to autophagy, further demonstrating deviation from the conventional
functions of ATG proteins (Martinez et al., 2015). LAP serves
as a host defense system that targets several pathogens, including Salmonella typhimurium and Aspergillus fumigatus (Huang
et al., 2009; Martinez et al., 2015).
In other cases, the growing list of noncanonical functions of ATG proteins has led the field to reevaluate a general
role of the autophagy pathway in certain aspects of microbial
pathogenesis. First, the autophagy pathway was proposed to
defend against Mycobacterium tuberculosis infection because
induction of autophagy by starvation or treatment with rapamycin reduces M. tuberculosis viability and because myeloidspecific deletion of ATG5 results in increased inflammation
and bacterial load in the lungs (Castillo et al., 2012; Watson et
al., 2012). Although this result originally assumed a protective
function of autophagy, recent work demonstrates that ATG5
is unique in its ability to restrict M. tuberculosis infection. In
stark contrast to ATG5, the genetic deletion of other essential
ATG genes (ATG3, ATG7, ATG12, ATG14L, and ATG16L) has
Published August 29, 2016
Acknowledgments
J. Debnath is supported by the National Institutes of Health
(CA126792, CA188404, and CA201849), U.S. Department of Defense Breast Cancer Research Program (W81XWH-11-1-0130), and
Samuel Waxman Cancer Research Foundation.
The authors declare no competing financial interests.
Submitted: 8 August 2016
Accepted: 8 August 2016
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Novel roles for ATG proteins in viral replication • Solvik and Debnath
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